FIGURE 4.

N-linked glycosylation in IL-1Rrp2 is required for IL-36R signaling. A, IL-1Rrp2 contains N-linked glycans. 293T cells were transfected with IL-1Rrp2 for 24 h, and the cell lysate was mock-treated or treated with PNGase F or O-glycosidase. The lysate was separated using SDS-PAGE, and IL-1Rrp2 was detected by Western blotting analysis. B, effects of tunicamycin treatment on NF-κΒ activation. 293T cells were transfected with IL-1Rrp2 for 6 h, and treated with increasing concentrations of tunicamycin for an additional 18 h, followed by stimulation with IL-36γ. The data were plotted as NF-κB fold induction relative to mock-treated sample. The IL-1Rrp2 level in the cell lysate was determined by Western blotting analysis. C, effects of swainsonine on NF-κB activation. D, effects of PNGase F or O-glycosidase treatment on the migration of endogenous IL-1Rrp2 in NCI cells. The level of IL-1Rrp2 in the cell lysate was detected by Western blotting analysis. E, effects of tunicamycin on IL-36R signaling in NCI cells. NCI cells treated with increasing concentrations of tunicamycin for 24 h were stimulated with IL-36γ. The IL-6 level in the medium (solid line) was determined by ELISA. Cell proliferation (dashed line) was assessed by WST-1 assay according to the manufacturer's protocol. IL-1Rrp2 accumulation was detected by Western blotting analysis. F, effects of swainsonine on IL-36R signaling in NCI cells. The solid line and dashed line denoted IL-6 level and cell proliferation, respectively. The Western blotting analysis showed the effects of swainsonine on IL-1Rrp2 accumulation.